The invention relates to the identification of protease inhibitors.
An undesirable level of protease activity occurs in a variety of conditions, including rheumatoid arthritis and other autoimmune diseases, tumor invasion, inflammation, and diseases caused by infections agents which produce proteases, such as HIV, rhinoviruses, hepatitis viruses, and herpes viruses. Protease inhibitors, particularly specific inhibitors, can be used to treat such conditions.
Thus, there is a need in the art for convenient and rapid methods of identifying protease inhibitors.
It is an object of the present invention to provide methods for identifying protease inhibitors. This and other objects of the invention are provided by one or more of the embodiments described below.
One embodiment of the invention is a method of screening a test substance for the ability to inhibit a protease having a proteolytic activity. The method encompasses the steps of contacting a target cell comprising a protease having a proteolytic activity with a viral display package in the presence of a test substance, wherein the viral display package comprises (a) a transferable label and (b) a recombinant viral envelope protein comprising in sequential order (i) a receptor-binding polypeptide which binds to a receptor on the surface of the target cell, (ii) a protease cleavage site for the protease expressed by the target cell, and (iii) a fusion-mediating polypeptide, such that proteolytic cleavage of the cleavage site does not permit substantial transfer of the transferable label from the viral display package to the target cell; and detecting the transferable label, if any, in the target cell, wherein detection of transferable label or a greater amount of transferable label in the target cell in the presence of the test substance relative to the absence of the test substance identifies the test substance as a protease inhibitor.
A xe2x80x9cproteasexe2x80x9d, also called an endoprotease, is an enzyme which hydrolyzes a peptide bond between a pair of amino acids located in a polypeptide chain. The proteolytic activity of the protease does not permit substantial transfer of the transferable label from the viral display package to the target cell.
The xe2x80x9cviral display packagexe2x80x9d comprises a transferable label and a recombinant viral envelope protein. The xe2x80x9ctransferable labelxe2x80x9d is a label whose presence is detectable in the target cell as a result of fusion of the viral display package and the target cell membrane. Thus, if the protease produced by the target cell cleaves the protease cleavage site in the recombinant envelope protein, the virus display package does not transfer label to the target cell. Alternatively, where the protease is inhibited, it is unable to cleave the cleavage site, and the virus display package can infect the target cell and transfer label.
Preferably, the transferable label is a gene encoding a selectable marker or is a reporter gene which encodes a detectable product.
The xe2x80x9csequential orderxe2x80x9d of components comprising a recombinant viral envelope protein occurs sequentially from the N- to C-terminus.
The xe2x80x9creceptor-binding polypeptidexe2x80x9d is capable of binding to a cognate receptor on the surface of a target cell, and thereby initiating gene delivery to the target cell. For example, the receptor binding polypeptide is capable of binding to a cognate receptor on a target cell, such as a viral envelope receptor on the surface of the target cell. The receptor-binding polypeptide should minimally include the receptor-binding domain of a protein, i.e., the portion of the protein that retains the ability to bind to the receptor on the cell. Thus, receptor-binding polypeptide (e.g., a displayed polypeptide) may be a receptor-binding domain of a viral envelope protein, such as a 4070A envelope protein or Moloney murine leukemia virus envelope protein, which (a) binds to a cell-surface receptor and (b) initiates gene delivery thereby. The underlying envelope protein (on which the receptor binding polypeptide is displayed) must retain its membrane fusion ability but does not necessarily need to retain its receptor-binding capability.
Certain alterations, such as mutations, deletions, or additions, can be made to the receptor-binding polypeptide which do not significantly affect its functions, as described above (a) and (b). One example of a receptor-binding domain useful in the context of this invention is the binding domain for the Pit-2/Ram-1 receptor of the 4070A virus. Other receptor-binding domains include the CAT-1 receptor-binding domain of the Moloney murine leukemia virus, GALV receptor-binding domain, or any of those from MLV envelope proteins.
The xe2x80x9cfusion-mediating polypeptidexe2x80x9d may be a fusion-mediating domain of a viral envelope protein or a substantially intact viral envelope protein and can be derived either from the same type of viral envelope protein as the receptor-binding polypeptide or from a different viral envelope protein. Viruses such as murine leukemia viruses (e.g., Moloney murine leukemia virus or 4070A murine leukemia virus) are particularly useful as sources of viral envelope proteins. In another embodiment, the recombinant viral envelope protein is a viral envelope protein of a murine leukemia virus, such as a Moloney murine leukemia virus or a 4070A, in which a protease cleavage site has been inserted between the receptor-binding and the fusion-mediating polypeptides.
The xe2x80x9ctransferable label,xe2x80x9d if any, is detected in the target cell. Detection of the transferable label or of a greater amount of transferable label in the target cell in the presence of the test substance relative to the absence of the test substance identifies the test substance as a potential protease inhibitor. The xe2x80x9camountxe2x80x9d of label transferred in the presence of an inhibitor is considered a xe2x80x9cgreater amountxe2x80x9d if it is at least 10, 20, 25, 50, 75, 85, 90, 95, 98, 99, or 100%, or at least 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold greater than the amount of transferable label that is transferred from the viral display package to a target cell in the absence of the protease inhibitor. If no amount of label is transferred in the absence of the protease inhibitor, then any detectable label in the presence of the inhibitor is indicative of inhibition and is a xe2x80x9cgreater amount.xe2x80x9d
A xe2x80x9cprotease cleavage sitexe2x80x9d according to the invention is a contiguous sequence of amino acids connected by peptide bonds which contains a pair of amino acids which is connected by a peptide bond that is hydrolyzed by a particular protease. Optionally, a protease cleavage site according to the invention includes one or more amino acids on either side of the peptide bond to be hydrolyzed, to which the catalytic site of the protease also binds (Schecter and Berger, Biochem. Biophys. Res. Commun. 27, 157-62, 1967). Thus, the protease xe2x80x9ccleavage sitexe2x80x9d also may include a protease recognition site, and both sites may be included within an amino acid linker sequence.
Alternatively, a xe2x80x9cpluralityxe2x80x9d of target cells (i.e. more than one target cell) is contacted with a plurality of viral display packages (i.e. more than one viral display package) in the presence of a test substance. In this case, detection of transferable label in a number of target cells or in a greater number of target cells in the presence of the test substance relative to the absence of the test substance identifies the test substance as a potential protease inhibitor. A xe2x80x9cgreater numberxe2x80x9d of target cells in which the transferable label is detected is any number of target cells which is at least 1, 2, 5, 10, 20, 50, or 100 more than the number of target cells in which the transferable label is detected in the absence of a protease inhibitor.
In a specific embodiment, an otherwise intact envelope protein has a protease cleavage site inserted at a position that abrogates the function of the envelope protein when cleaved.
In another embodiment, the invention encompasses an MLV envelope protein which is capable of binding to a target receptor and subsequently causing virus-cell fusion, and containing a heterologous protease cleavage site, such that the normal functions of the envelope protein are not impaired. Upon cleavage at the introduced site by a protease, the envelope protein no longer functions.
Another embodiment of the invention is a method of delivering a transferable label to a target cell. A target cell comprising a protease having a proteolytic activity is contacted with a viral display package and a protease inhibitor. The viral display package comprises a transferable label, preferably an expressible polynucleotide, and a recombinant viral envelope protein. The recombinant viral envelope protein comprises in sequential order (I) a receptor-binding polypeptide which binds to an amphotropic receptor on the surface of the target cell, (ii) a protease cleavage site for the protease and (iii) a fusion-mediating polypeptide which binds to an ecotropic receptor on the surface of the target cell.
The receptor-binding polypeptide is preferably a receptor-binding domain of an amphotropic viral envelope protein (4070A). The fusion-mediating polypeptide is preferably a fusion-mediating domain of an ecotropic viral envelope protein, such as a Moloney murine leukemia virus envelope protein.
The target cell is a eukaryotic cell, preferably a mammalian cell, even more preferably a human cell. The target cell can be present in a mammalian or human body or can be in vitro.
In a preferred embodiment, the target cell expresses amphotropic virus receptors, but does not express ecotropic virus receptors, for example, an xe2x80x98unmodifiedxe2x80x99 human cell line. Cells such as lymphocytes and bone marrow cells are therefore useful according to the invention, provided they are transfected or subject to infection with a retrovirus, e.g., GALV.
Transfer of the transferable label from the viral display package to the target cell is dependent upon inhibition of the proteolytic activity of the protease produced by the target cell. A greater amount of transferable label is thereby delivered to the target cell in the presence of the protease inhibitor relative to the absence of the protease inhibitor. Alternatively, a plurality of target cells are contacted with a plurality of viral display packages and a protease inhibitor. In this case, the transferable label is delivered to a number of target cells, or to a greater number of target cells, in the presence of the protease inhibitor relative to the absence of the protease inhibitor.
Thus, the present invention provides an innovative approach to the identification of protease inhibitors, as well as the delivery of transferable labels to a target cell in the presence of a protease inhibitor.